The application of genetic modification offers considerable opportunities for more efficient and more effective fish aquaculture. Susceptibility to infectious diseases and limitations in growth rates could be successfully tackled by the application of transgenic technology. Acceptance of transgenic technology applied to commercial fish has to surmount technical difficulties, regulatory barriers and public acceptance concerns. It seems desirable to implement transgenesis strategies that cause the least qualitative and quantitative alterations in the genetic composition of the resulting enhanced fish. With Atlantic salmon being the most important farmed fish worldwide, it seemed of interest to obtain genomic sequences from this species capable of directing autogenic expression of selected transgenes. We now report the isolation of the β-actin promoter from genomic DNA of Atlantic salmon locally produced in Chile. The activity of the promoter was demonstrated ex vivo in cultured salmon cells, as well as in vivo in transgenic fish. Transiently transfected CHSE-214 cells exhibited constitutive expression of reporter constructs driven by the cloned salmon β-actin genomic region. The suitability of the Atlantic salmon β-actin promoter to drive expression of the fluorescent protein EGFP in transgenic zebrafish was demonstrated. Germline transmission of the transgenes suggests that this promoter could be safely applied into fish transgenesis without health concerns. The isolation of functionally competent β-actin promoter from Atlantic salmon opens the way for its use in autogenic transgenesis in this commercially important salmonid species.